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Beatrice Yue, Hongyu Ying, Sanja Turturro, Xiang Shen, Tara Nguyen, Ruth Zelkha; Viral Expression of Wild-Type and Mutant Optineurin in Rats. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2443.
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To determine whether impairment of the ubiquitin-proteasome system and induction of autophagy observed previously in vitro occur in vivo and assess whether the accumulated mutant optineurin can be cleared by autophagic enhancer rapamycin. Optineurin is a gene associated with normal tension glaucoma and amyotrophic lateral sclerosis.
Adeno-associated type 2 viral (AAV2) vectors for green fluorescence protein (GFP), GFP-tagged wild-type and E50K optineurin were intravitreally injected into rats for expression in retinal ganglion cells (RGCs). Phosphate buffered saline (PBS) was injected in parallel into rat eyes as controls. The intraocular pressure (IOP) was monitored 2, 3, and 5 weeks; and animals were euthanized 5 weeks following the injection. Rat eyes were enucleated and retinal flat mounts were prepared. GFP-positive RGCs were counted. The levels of optineurin, proteasome subunit β type 5 (PSMB5), an indicator of the proteasome activity, and LC3, an autophagic marker, in the retina were evaluated by immunofluorescence and Western blotting. The optic nerve heads were fixed and embedded in Epon resin. Thin sections were stained with toluidine blue and the number of axons was determined. To investigate the effects of rapamycin, two sets of rats were injected with GFP or E50K-GFP viral vectors. One set of animals received rapamycin (20 mg/Kg body weight) via intraperitoneal injections 3 times a week for 5 weeks. The rats were subsequently sacrificed and the eyes were examined as above.
Rat eyes that received wild-type and E50K optineurin vectors demonstrated RGC and axonal loss in comparison to GFP and PBS controls. The IOP remained un-elevated. By immunostaining and immunoblotting, the level of PSMB5 was found reduced and that of LC3 was increased, indicating that the ubiqutin-proteasome system was compromised and the autophagy process was provoked. After rapamycin treatment, AAV2-E50K-GFP-injected rats showed normal RGC and axonal counts. The levels of optineurin and PSMB5 also returned to the normal range. It appeared that the accumulated mutant optineurin could be cleared by rapamycin.
The ubiquitin-proteasomal activity was reduced and autophagy was enhanced when wild-type and E50K optineurin was overexpressed in rat eyes. This study confirmed and validated previous in vitro findings. Rapamycin was effective in rescuing the E50K optineurin-mediated RGC and axonal loss in the in vivo rat model.
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